Biological Characterizations Of The Mutant Strain With Hor2 Gene Deleted In Sacchromyces Cerevisiae

Since the industrial revolution, the development of international economic growth has not been able to deviate from the development of energy any longer.The fossil fuel is the mainstream of energy supply,the backbone of economic growth all along. With the development of technology,the progess of industry and the expansion of the world's population,however, the demand for energy sources has become more and more pressing.In addition,the fossil fuel is a kind of unrenewable fuel,which will result in the environmental pollution in a sense therefore,it will be a main point for human to find out a renewable fuel. Bio-renewable energy sources have been paid great attention; thereinto, the fuel ethanol,a bio-renewable fuel,is especially important because of its advantages. As the eukaryotic model organism, Saccharomyces cerevisiae has been diffusely used in the ethanol fermentation in these days.Glycerol is one of the main by-products in alcohol metabolism in Saccharomyces cerevisiae. There are two genes (HOR2, RHR2) that encode isoforms of glycerol 3-phosphatase involved in Glycerol metabolism in Saccharomyces cerevisiae. HOR2p is mainly involved in the Cellular Responses to osmotic stress,while RHR2p mainly involved in the Cellular Responses to anerobic.Therefore,the deletion of HOR2 or RHR2 (here,we take HOR2) can interrupt or partially interrupt the Glycerol metabolism with the result that the glycerol is decreased.At last,there are more carbon sources which are transferred into alcohol,as will help to study the molecular regulatory mechanism of Saccharomyces cerevisiae.By making use of the same long oligonucleotides with 22 or 19 3'nucleotides complementary to sequences in the templates pUG6 and 45 5'nucleotides which annealed to sites upstream or downstream of the genomic target sequence that would be deleted,the gene disruption cassette is created with PCR.After that,the gene disruption cassette is transformed to the cells of Saccharomyces cerevisiae YS2. Then,the positive transformants are confirmed with PCR in order to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.As long as it is correctly integrated into the genome of YS2, the marker can be efficiently redeemed after transformating the pSH65 to YS2 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure with the result that the marker gene is removed and a single loxP site is left behind at the chromosomal locus.By repeating the above procedure the diploid mutant YS2-HOR2 is generated, which could enhance the output of ethanol with 1.96% and decrease the output of glycerol with 3.34% by shaking culture in flask compared with the original strain YS2...